Part:BBa_K4907138

I13453-B0034-ccdA-B0015

Biology

ccdA is another gene found within the ccd operon, encoding the antidote protein (CcdA) that protects cells from the toxic effects of CcdB.

Usage and design

To verify the role of CcdA in resisting the CcdB toxin, we designed a composite part: BBa_K4907138 to characterize ccdA. The constructed circuit was transformed into E. coli DH10β, followed by chloramphenicol selection of positive transformants, and confirmation was carried out through colony PCR and sequencing.

Fig. 1 Gene Circuit of BBa_K4907138

Characterization

Agarose gel electrophoresis (AGE)

In the construction of this circuit, colony PCR and gene sequencing were used to verify the correctness of the transformants. At around 689bp, a target band of approximately 750bp was observed (Fig. 2).

Fig. 2 DNA gel electrophoresis of the colony PCR products of BBa_K4907138_pSB1C3

Verification of dual-plasmid transformation

To validate the resistance of CcdA to ccdB, we performed a dual-plasmid transformation.

Table. 1 Performance of different dual-plasmid systems in E. coli DH10β

The results show that E. coli DH10β transformed with both toxins and antitoxins also exhibited good growth. This confirmed the killing effect of the toxin CcdB again and the neutralization of antitoxin CcdA.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 125
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 65
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]